This invention relates to maintenance of sperm viability to increase the success rate of artificial insemination (AI).
AI is now a fundamental technology for the intensive breeding of domestic animals, in human infertility treatments and in wildlife conservation programmes for the breeding of threatened species. Nevertheless, it has become clear that current semen preservation techniques severely compromise the sperm's survival in the female reproductive tract and hence limit the successful application of the technique.
Sperm survival is particularly compromised when spermatozoa cannot be delivered directly into the uterus because the cervical anatomy is too complex, for example in sheep. This significantly reduces the efficiency of AI. Large numbers of viable spermatozoa must be used to maximize the chance of fertilization, therefore making this technique uneconomical. Surgical intrauterine insemination by laparoscopy is an efficient way of solving this problem and through use of this method conception rates of 80% are now common in sheep and other species. However, this method increasingly is regarded as unacceptable for routine agricultural use on grounds of welfare; routine use of this surgical approach is expected to be curtailed within a relatively short period.
Means to improve the success rate of non-surgical methods is therefore urgently required. One means of achieving this will be by extending the lifespan of spermatozoa in the female reproductive tract.
Following mating (natural insemination), inseminated mammalian spermatozoa are transported to the oviduct where a reservoir of spermatozoa is formed. Studies in several species have shown that the reservoir is limited to the caudal isthmus. The spermatozoa are held in the isthmus until ovulation, when a small number are released to meet the egg(s). During storage in the isthmus, many spermatozoa attach to the oviductal epithelial cells. Attachment to oviductal epithelial cells is important in maintaining sperm viability both in vivo and in vitro. Spermatozoa attachment to oviductal epithelial cells is initiated by uncapacitated spermatozoa. The process of capacitation, along with the switch to the hyperactivated flagellar beating pattern, appears to coincide with the ability of spermatozoa to be released from the oviductal reservoir.
Coculture with whole oviductal epithelial cells in vitro improves the viability of sperm from a number of species including rabbit, cow, sheep, horse, pig and human. It seems this is a widespread characteristic of oviductal cells. However the mechanism by which oviductal cells maintain sperm viability is unknown. Both oviductal secretory products and direct membrane contact between spermatozoa and oviductal epithelial cell membranes have been reported to bestow this beneficial effect.
Many studies in the past have only investigated the role of oviductal secretory products (proteins) on spermatozoa.
Oviductal secretory products have been reported to improve the viability of sperm. These secreted proteins are present in oviduct fluid and the fluids from which they are derived are collected via indwelling cannulae in the ampulla and isthmus of the oviduct. These secreted proteins are not derivable from whole oviductal cells in vitro, but must be collected by cannulation of the oviduct of cycling animals.
Catalase is an example of a secretory protein; this enzyme is known to protect spermatozoa against damage by reactive oxygen species.
The inventors have shown previously that whole oviduct epithelial cells could be isolated and cultured, and that when co-incubated with spermatozoa at 39° C., the life of the spermatozoa could be extended for 2 to 3 days beyond the maximum lifespan of control spermatozoa incubated without cells. Sperm lifespan was judged by the use of tests for plasma membrane integrity.
The inventors have further shown that incubation of spermatozoa with porcine oviductal apical plasma membrane (APM) extends the life of the cultured spermatozoa.